Serum heme oxygenase‐1 level predicts clinical outcome after acute ischemic stroke

Abstract Aims The relationship between heme oxygenase‐1 (HO‐1) and human ischemic stroke outcome remains unclear, which was investigated in this study. Methods Acute ischemic stroke patients admitted within 24 h were enrolled. Serum HO‐1 levels at baseline were measured via ELISA. Poor 3‐month functional outcome was defined as modified Rankin Scale (mRS) score 3–6. Multivariable‐adjusted binary logistic regression and restricted cubic spline models were employed to examine association between serum HO‐1 and functional outcome. HO‐1's additive prognostic utility was assessed by net reclassification index (NRI) and integrated discrimination improvement (IDI). Results Of 194 eligible patients, 79 (40.7%) developed poor functional outcomes at 3‐month follow‐up. The highest quartile of serum HO‐1 was independently associated with a lower risk of poor functional outcome (adjusted OR 0.13, 95% CI 0.04–0.45; p = 0.001) compared with the lowest HO‐1 category. The relationship between higher HO‐1 levels and reduced risk of poor functional outcome was linear and dose responsive (p = 0.002 for linearity). Incorporating HO‐1 into the analysis with conventional factors significantly improved reclassification for poor functional outcomes (NRI = 41.2%, p = 0.004; IDI = 5.0%, p = 0.004). Conclusions Elevated serum HO‐1 levels at baseline were independently associated with improved 3‐month functional outcomes post‐ischemic stroke. Serum HO‐1 measurement may enhance outcome prediction beyond conventional clinical factors.

biomarkers holds promise for augmenting our ability to identify individuals at heightened risk of unfavorable prognosis. 5ring the occurrence of cerebral ischemia, the production of free radicals and ensuing oxidative stress emerge as a pivotal contributor to causing neuronal damage and subsequent cell demise. 6,7ven the compelling association between redox imbalance and ischemic injury, biomarkers affiliated with the endogenous antioxidant system may furnish valuable insights into prognosticating outcomes in individuals afflicted by cerebral infarction. 8,9This may open new vistas for therapeutic interventions and clinical decisions.A particularly well-investigated avenue revolves around the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, with heme oxygenase-1 (HO-1) serving as a central antioxidant enzyme. 8,10Moreover, a clinical study indicates that serum levels of HO-1 are elevated after hemorrhagic strokes, 11 and that HO-1 levels are higher in ischemic strokes than in transient ischemic attacks. 12e intracellular enzyme HO-1 facilitates the degradation of heme into carbon monoxide (CO), biliverdin (BV), and ferrous iron (Fe 2+ ), thereby effectively mitigating oxidative stress. 13,14Prior research has demonstrated that transgenic mice overexpressing HO-1 evince significantly diminished infarct volumes in the wake of irreversible occlusion of the middle cerebral artery. 15Additionally, therapy with an adenoviral vector augmenting HO-1 expression engenders reduced infarct volumes and ameliorated neurological deficits in experimental models of brain ischemia, underscoring its neuroprotective potential. 16Nevertheless, scant attention has been devoted to exploring the correlation between blood HO-1 levels and clinical outcomes in stroke patients.Consequently, this study aimed to investigate whether blood HO-1 levels correlate with poor functional outcomes after acute ischemic stroke.

| ME THODS
This study was performed according to the Strengthening the Reporting of Observational Studies in Epidemiology reporting guidelines 17 and approved by the Ethics Committee on Biomedical Research, West China Hospital of Sichuan University, which conformed to the recommendations of the Declaration of Helsinki.All patients or their relatives provided written informed consent.

| Study design and participants
This cohort study was conducted using data from the Chengdu Stroke Registry, an ongoing registry that has prospectively and consecutively enrolled patients with ischemic stroke who were admitted to the Department of Neurology in West China Hospital from 2002.The registry has previously been described in detail. 18S patients admitted and recorded in the registry, from December 2018 to December 2021, were retrospectively included if they (1)   were at least 18 years of age; (2) were admitted to the hospital within 24 h of stroke onset; (3) had a confirmed diagnosis of ischemic stroke based on computed tomography (CT) or magnetic resonance imaging (MRI); (4) had blood samples available for serum HO-1 measurements; (5) had a modified Rankin Scale (mRS) score of 0-2 prior to stroke; and (6) had completed 3-month follow-up.In accordance with the World Health Organization (WHO) criteria, ischemic stroke is defined as acute focal neurological dysfunction caused by single or multiple sites of the brain or retina infarction lasting more than 24 h, or with evidence of acute infarction based on neuroimaging or other technique in the clinically relevant area of the brain. 19Patients were excluded if they had (1) major comorbidities or late-stage diseases, including severe liver disease, heart failure, end-stage kidney disease, serious infection, malignant tumors, etc.; and (2) concurrent autoimmune diseases.

| Data collection
Details of patient demographics, onset-to-admission interval, stroke severity on admission, stroke etiology, and vascular risk factors were recorded.The National Institutes of Health Stroke Scale (NIHSS) score was used to assess stroke severity. 20Atrial fibrillation (AF) was defined as a history of persistent or paroxysmal AF, confirmed by previous electrocardiogram (ECG) or diagnosed by physician based on an ECG and/or 24-h ECG monitoring during hospitalization. 21Other vascular risk factors included diabetes, hypertension, hyperlipidemia, coronary artery disease, current smoking, alcohol consumption, and history of stroke, which have been described in previous studies. 22,23Stroke etiology was classified according to Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria. 24Acute reperfusion therapy, such as intravenous thrombolysis (IVT) and/or endovascular therapy (EVT), was also recorded.

| Serum HO-1 measurement
Peripheral blood samples were collected within 48 h of the patient's arrival at the Emergency Department.Blood samples were centrifuged at clinical laboratories of West China Hospital and then serum samples were immediately frozen at −80°C.Serum concentrations of HO-1 were detected by laboratory technicians blinded to the clinical data and outcomes of the participants and using enzymelinked immunosorbent assay (ELISA) with a commercially available ELISA kit (catalog: MB-1508A; Jiangsu Meibiao Biotechnology Co., Ltd., Jiangsu, China) in the laboratory of the K J Biotechnology Co., Ltd, Sichuan, China.

| Outcome assessment
The mRS was prospectively assessed at 3 months after stroke onset through telephone interviews with patients or family members by well-trained neurologists.A poor functional outcome at 3 months was defined as an mRS score of 3-6. 25

| Statistical analysis
Categorical variables are expressed as counts with percentages, while continuous data are presented as medians accompanied by their interquartile ranges (IQRs).Participants were grouped according to quartiles of HO-1 levels.Linear trends in baseline characteristics across quartiles of HO-1 levels were tested by Cochran-Armitage trend χ 2 test for categorical variables and generalized linear regression analysis for continuous variables.Univariable and multivariable binary logistic regression models were used to determine the independent associations of the clinical outcomes with HO-1 quartiles and HO-1 concentration as a continuous variable.Multivariable ordinal logistic regression was used to assess the association between mRS scores and HO-1 quartiles.Variables with a potential association with the outcome (p < 0.10) from univariate analysis were analyzed via multivariable models.Crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated.To test for linear trend, a term with the median value of each quartile of HO-1 was entered into the model as a continuous variable.
In addition, the R package rcssci V.0.4.0 was used to generate restricted cubic spline fitted to a logistic regression model in order to provide more precise estimates and explore the shape of the association between serum HO-1 levels and the clinical outcome. 26Three knots for spline were placed at the 10th, 50th, and 90th percentiles of HO-1, with the 10th percentile serving as a reference point.
To validate the robustness of the association between continuous HO-1 concentration and poor functional outcome, a subgroup analysis was conducted while adjusting for the aforementioned covariates.Potential interactions between HO-1 and subgroup variables were probed through likelihood-ratio tests of models encompassing interaction terms.Furthermore, the predictive performance of serum HO-1 as a continuous variable for the clinical outcome was assessed.
Predictive models were constructed using either conventional clinical factors alone as the basic model (containing clinical variables with p < 0.10 in univariate analyses which were adjusted in the multivariate logistic regression model), or the HO-1 level combined with these factors.To evaluate the calibration of predictive models, the Hosmer-Lemeshow χ 2 statistic was used.Net reclassification index (NRI) and integrated discrimination improvement (IDI) were calculated to assess improvements in risk reclassification of model with additive HO-1 compared with the basic model. 27l statistical analyses and graphics were generated using SPSS 24.0 (IBM, New York, USA), R 4.3.0(the R project for statistical computing), and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA).All P values were two-tailed tests with a statistical significance level of 0.05.

| Baseline characteristics
The initial sample consisted of 1363 consecutive AIS patients admitted to our institution within 24 h from December 2018 to December 2021.A total of 194 patients were included in the present study after the exclusion of patients who did not fulfill the selection criteria (Figure 1).There was no significant difference between the baseline characteristics of those included and excluded, except for the stroke onset-to-admission interval and alcohol consumption (Table S1).
Among 194 included patients, the median age was 66 years, the male proportion was 63.9%, and the median baseline HO-1 concentration was 28.11 ng/mL (25th and 75th percentiles of 24.05 and 31.03ng/ mL, respectively).In general, higher levels of HO-1 were associated with younger age, male sex, and lower baseline NIHSS scores (all p trends < 0.05; Table 1).Lower HO-1 concentrations were associated with higher proportions of hypertension, AF, and stroke history, and lower proportions of current smoking and alcohol consumption (all p trends < 0.05).More patients with lower HO-1 concentrations received EVT (p trend = 0.009).
F I G U R E 1 Flow chart for patient screening.

| Serum HO-1 levels and poor functional outcome at 3 months
Seventy-nine patients (40.7%) had poor functional outcomes at 3 months.Serum HO-1 levels were significantly lower in patients with versus without poor functional outcomes (25.72 vs. 29.74ng/ mL, p < 0.001; Figure 2A).A stepwise decrease in the risk of poor functional outcome was observed with higher quartiles of HO-1 (p < 0.001; Figure 2B).Univariable binary logistic regression analysis also showed that a higher HO-1 level, analyzed either as a continuous or ordinal (in quartiles) variable, was associated with a lower risk of poor functional outcome (all p < 0.05; Table S2).The highest quartile (compared with the lowest quartile) of serum HO-1 was significantly, independently associated with a lower risk of poor functional outcome (adjusted OR 0.13, 95% CI 0.04-0.45;p = 0.001; p trend < 0.001), after adjusting for age, sex, NIHSS, AF, alcohol consumption, current smoking, TOAST, and reperfusion therapy (Table 2, Figure S1 and Table S3).Moreover, an elevated serum HO-1 level was also independently associated with a decreased risk of poor functional outcome, when analyzed as a continuous variable (adjusted OR 0.88, 95% CI 0.81-0.95,p = 0.001; Table 2).Furthermore, multivariable-adjusted restricted cubic spline analysis demonstrated an inverse linear relationship between serum HO-1 levels and poor functional outcomes (p = 0.002 for linearity; Figure 3).In addition, there was an inverse dose-response relationship between HO-1 levels and mRS score at 3 months after ischemic stroke (p for trend = 0.004; Figure S2).

TA B L E 1
Baseline characteristics of participants according to quartiles of HO-1.

| Subgroup analysis of the association between serum HO-1 and poor functional outcome
According to the subgroup analyses stratified according to age, sex, hypertension, AF, admission NIHSS score, smoking, alcohol consumption, stroke etiology, and acute reperfusion treatment (with or without IVT and/or EVT), higher serum HO-1 levels were significantly associated with decreased risk of poor functional outcome in most subgroups (Figure S3).There was no significant interaction between HO-1 and these stratified factors (all p for interaction > 0.05).

| DISCUSS ION
Despite extensive basic research demonstrating HO-1's protective effects against cerebrovascular diseases, 15,28 data on its impact on clinical outcomes in patients with acute ischemic stroke have been scarce.Therefore, our study explored the relationship between serum HO-1 levels and the clinical outcome of stroke patients based on data from an ongoing prospective hospital-based stroke registry.We found higher serum HO-1 levels independently associated with a lower risk of poor 3-month functional outcome after an acute ischemic stroke.This association remained consistent across various subgroups, highlighting the robustness of our findings.Notably, the relationship between higher HO-1 levels and lower risk of poor functional outcome appeared to be linear and dose responsive.
Furthermore, incorporating HO-1 into a predictive model alongside conventional clinical factors significantly improved risk prediction for poor functional outcomes.Our findings shed light on the significant role of HO-1 in ischemic stroke outcomes and its potential as a prognostic biomarker.
Several previous studies have provided insights into the role of HO-1 in the context of ischemic stroke.For instance, genetic variations in the HO-1 gene promoter, leading to increased HO-1 expression, were associated with a reduced risk of ischemic cerebrovascular events in Austrian patients with normal plasma lipid levels. 29Additionally, shorter HO-1 promoter genotypes were linked to a decreased risk of ischemic stroke, particularly in Chinese individuals with low levels of high-density lipoprotein cholesterol. 30Another study focused on atherosclerotic stroke patients and explored the correlation of HO-1 gene polymorphisms with clinical prognosis in an Asian cohort, suggesting that patients carrying at least one A allele showed significantly better outcomes compared to those with a TT genotype, potentially attributable to the heightened expression of HO-1. 31Taken together, these findings suggested that HO-1 level may be related to outcomes after ischemic stroke; however, none of these studies specifically investigated the prognostic role of HO-1 levels in stroke patients.In addition, while two studies examined the association between HO-1 levels in the cerebrospinal fluid and functional outcomes in patients with subarachnoid hemorrhage, conflicting findings were found.One study found that higher levels of HO-1 mRNA were associated with favorable functional outcomes, 32 whereas in another study, higher levels of HO-1 protein were associated with more unfavorable outcomes. 33Our study attempted Previous literature suggests that HO-1 may protect ischemic stroke through its anti-inflammatory, antioxidant, antiapoptotic, and vasorelaxant effects by degrading the oxidant heme. 8Experimental models simulating brain ischemia demonstrated that augmenting HO-1 expression through gene therapy led to reduced infarct volume and alleviation of neurological impairments. 16,34Additionally, HO-1's downstream metabolites may also exert a protective effect in ischemic stroke.CO exerts a potent anti-inflammatory effect in diverse diseases, and biliverdin is subsequently transformed into bilirubin, 35 which can function as an antioxidant.Both of these mechanisms may contribute to the protective role of HO-1 in cerebral ischemia. 36,37ere were several limitations to the present study.First, due to the retrospective selection of eligible patients, we had to exclude a considerable number of acute stroke patients without blood samples for HO-1 measurement.Nevertheless, the large comparability of baseline characteristics between included and excluded patients suggests negligible selection bias.Second, only baseline serum HO-1 levels were measured, while early complications after an index TA B L E 2 The independent association between HO-1 levels and clinical outcome after stroke adjusted by multivariable binary logistic regression.

| CON CLUS ION
Our study highlights the independent association between higher serum HO-1 levels on baseline and a reduced risk of poor functional HO-1 levels according to the clinical outcome.Decreased serum HO-1 was associated with poor 3-month functional outcomes after stroke.(A) Lower HO-1 level in patients with (median 25.72 ng/mL [IQR 21.06-29.41ng/mL]) versus without poor functional outcome at 3 months (29.74 ng/mL [IQR 26.32-31.76ng/mL]).Mann-Whitney U Test, **p < 0.001.Box plot represents the median and IQR, and whiskers the 10th and 90th percentile.(B) Lower rates of poor functional outcome with higher HO-1 quartiles.Chi-squared test, **p < 0.001.HO-1, heme oxygenase-1; HT, hemorrhagic transformation; IQR, interquartile range.touncover the relationship between serum HO-1 and long-term outcomes after stroke, and we found that serum HO-1 levels are inversely related to poor functional outcomes after 3 months, suggesting the possibility of HO-1 acting as a neuroprotective target in stroke recovery.Moreover, we demonstrated that the inclusion of serum HO-1 levels in predictive models could enhance risk prediction for acute ischemic stroke outcomes.

F I G U R E 3 | 7 of 8 WANG
Relationship between HO-1 levels and clinical outcome of patients with acute ischemic stroke.ORs and 95% CIs derived from restricted cubic spline regression, with knots placed at the 10th, 50th, and 90th percentiles of the distribution of HO-1 levels.The reference point is the 10th percentile (20.75 ng/mL).A significant inverse linear relationship between serum HO-1 levels and poor functional outcome at 3 months (p = 0.002 for linearity).ORs were adjusted for age, sex, National Institutes of Health Stroke Scale, atrial fibrillation, current smoking, alcohol consumption, and the Trial of ORG 10172 in Acute Stroke Treatment classification and reperfusion therapy.CI, confidence interval; HO-1, heme oxygenase-1; OR, odds ratio.et al. stroke or other conditions may affect the HO-1 levels; hence, serial measurements could potentially provide more comprehensive prognostic information.Future studies are needed to investigate the associations between dynamic variations in serum HO-1 levels and the prognosis of ischemic stroke.Third, the possibility of residual confounding factors remains, as treatments beyond acute reperfusion therapies were not considered in our multivariate analyses.Finally, our study cohort comprised solely Chinese patients, warranting caution when generalizing the findings to other populations.Therefore, further large-scale prospective studies conducted among different populations are needed to replicate our findings.

outcome at 3 TA B L E 3
months following ischemic stroke.Serum HO-1 levels demonstrate potential as a prognostic biomarker for acute ischemic stroke outcome, offering additive prognostic value when integrated with conventional clinical factors.To validate and extend our findings, further prospective studies with larger and more diverse samples, serial assessments of HO-1 levels post-stroke, and examination in different populations are warranted.Calibration and risk reclassification of models predicting clinical outcome of acute ischemic stroke, with or without HO-1.

HO-1 (ng/mL) p trend HO-1 level as a continuous variable (ng/mL) <24.05 24.05-28.11 28.11-31.03 ≥31.03
Note: Model adjusted for age, sex, National Institutes of Health Stroke Scale, atrial fibrillation, current smoking, alcohol consumption, and the Trial of ORG 10172 in Acute Stroke Treatment classification and reperfusion therapy.
Note: Basic model included age, sex, National Institutes of Health Stroke Scale, atrial fibrillation, current smoking, alcohol consumption, and the Trial of ORG 10172 in Acute Stroke Treatment classification and reperfusion therapy.Abbreviations: CI, confidence interval; HO-1, heme oxygenase-1; IDI, integrated discrimination improvement; mRS, modified Rankin Scale; NRI, net reclassification index; Ref, reference.
a p value < 0.05.